The consumers prefer to buy fresh bread and they want it to remain fresh for a long time. Retarding the staling has always been a challenge to producers of bakery ingredients. The fact that the production of bread is more and more centralised and farther away from the distribution points puts an even larger pressure on the development of additives and ingredients to maintain the softness of bread. Also soft rolls, hamburger, buns and pastry products are subject to staling and a loss of softness. There are a number of ingredients known to retard the staling of bread and soft bakery products. Fat and emulsifiers such as distilled monoglycerides and stearoyllactylates are already used since decades. Mono-, di- and polysaccharides have a positive influence on water retention and binding. Water loss is often associated with staling and the saccharides have positive influence on the mouthfeel of baked products and thus diminish the perception of staling. Amylases are known to have a beneficial effect on staling and starch retrogradation.
Bread staling is a complex phenomenon. It is perceived as a softening of the crust, a hardening of the crumb and the disappearance of fresh bread flavour. The hardening of the crumb is not only due to a loss of water during storage as was already demonstrated by Boussingault in ((1852) Ann. Chim. Phys. 3, 36, 490). It is the result of a number of physico-chemical processes. Over the years, researchers have tried to unravel these processes and developed different theories.
In the early days, bread firming was attributed solely to the retrogradation of starch (Katz, J. R. (1930) Z. Phys. Chem., 150, 37-59). It was shown by X-ray diffraction that the starch in bread is forming a micro-crystalline structure during storage. Later it was shown that the water soluble starch fraction diminished during bread staling (Schoch et al. (1947) Cereal Chem., 24, 231-249), which concludes that during baking starch granules absorb water. The linear amylose chains become soluble and diffuse to the water phase. In time more and more amylose is present in the water phase. So the amylose is partially leached out of the swollen starch granules. The branched amylopectine remains in the granules. The leaching process is limited by the available water. During cooling the amylose retrogrades very quickly and forms a gel. The retrogradation of amylopectine is believed to involve primarily association of its outer branches and requires a longer time than does the retrogradation of amylose, giving it prominence in the staling process, which occurs over time after the product has cooled, aggregate more slowly, due to stereochemical interferences. The amylopectine formed intramolecular bonds. The prominent role of starch in staling of bread is further illustrated by the use of carbohydrases to diminish or to slow down the staling of baked products. It was shown (Conn J. F. et al. (1950) Cereal Chem., 27, 191-205) that amylases from bacterial or fungal origin slow down the rate of staling of bread and result in a less firm crumb structure. The addition of thermostable alfa-amylases or beta-amylases is most effective. However this also results in a gummy and sticky crumb.
The document EP0412607 discloses the use of a thermostable alfa-1,6-endoglucanase or an alfa-1,4-exoglucanase to reduce staling; EP0234858 discloses the use of a thermostable maltogenic beta-amylase to retain the crumb softness.
However, it is still not clear whether the anti-staling effect is due to the dextrins produced or to the modification of the amylose and amylopectine and the consequent reduced tendency to crystallise. Also the influence of emulsifiers as glycerolmonostearate and sodiumstearoyllactylate seems to confirm the role of starch in bread crumb firming (Schuster G. (1985) Emulgatoren für Lebensmittel—Springer Verlag 323-329). It is the interaction between these emulsifiers and the starch which results in a changed starch conformation that accounts for the observed reduction of staling.
As there was not always a good correlation between starch structure and staling (Zobel H. F. et al (1959) Cereal Chem., 36, 441), other flour constituents were also investigated. The role of flour proteins in the crumb firming process has been studied but it was found that they were less important than starch (Cluskey, J. E. (1959) Cereal Chem., 36, 236-246.), (Dragsdorf, R. D. et al. (1980) Cereal Chem., 57, 310-314) studied the water migration between starch and gluten during bread storage. These authors concluded that due to a change in the cristallinity of the starch, it adsorbed more water, so the water migrates from the gluten to the starch and so less free water is available.
In later study (Martin et al. (1991) Cereal Chem., 68(5), 498-503 and 503-507), it appears that the high molecular weight dextrins do not have an antifirming effect on bread crumb. Instead, the high DP dextrins may entangle and/or form a hydrogen bond with protein fibrils, thus effectively cross-linking the gluten. Consequently, the firming rate is increased. It is stated that in weaker flours the gluten interacts stronger with the starch granules. This results in bread crumb that firms faster. However better gluten quality and stronger flour also result in higher loaf volume and thus in a softer crumb. Axford et al. (1968) cited in Faridi, H. (1985) Rheology of wheat products, AACC, p. 263-264) showed that the loaf specific volume was a major factor in measuring both the rate and the extent of firming. So the role of gluten in bread firming remains still questionable and few attempts have been made to slow down firming based on gluten modification.
Proteases have a long history of use in the baking sector. They are mostly used by the baker to reduce mechanical dough development requirements of unusually strong or tough gluten. They lower the viscosity and increase the extensibility of the dough. In the end product they improve the texture compressibility, the loaf volume and the bread colour. Also the flavour can be enhanced by production of certain peptides. The proteases mellow the gluten enzymatically rather than mechanically. They reduce the consistency of the dough, decreasing the farinograph value. The proteases most used in baking are from Aspergillus oryzae and Bacillus subtilis. The neutral bacterial proteases are by far more active on gluten than the alkaline proteases. Papain, bromelain and ficin are thiol-proteases extracted from papaya, pineapple and figs. Especially papain is very reactive towards gluten proteins. Bacterial proteases and papain, especially neutral proteases, are used in cookies, breadsticks and crackers where a pronounced slackening of the dough is wanted. However, in breadmaking, a more mild hydrolysis of fungal proteases is preferred.
Proteases also have major disadvantages. The action of the proteases is not limited in time, it continues after mixing and weakens the dough structure in time. This phenomenon increases the risk of weakening the dough and increases the stickiness of the dough. Sometimes their action is even enhanced by the pH drop during fermentation. The use of proteases in baking requires strict control of the bulk fermentation and proofing conditions of the dough. The proteases are inactivated during baking (Kruger, J. E. (1987) Enzymes and their role in cereal technology AACC 290-304). Especially neutral Bacillus proteases and papain should be dosed very carefully as overdoses slacken the dough too much. This may result in dough collapse before ovening or a lower bread volume and a more open crumb structure. Especially in Europe, where the flours are weaker than in the US or Canada, the risk of overdosing protease is very present.
Furthermore, proteases also increase stickiness because by the hydrolytic action water is released from the gluten (Schwimmer, S. (1981) Source book of food enzymology-AVI Publishing, 583-584). This means that in practice proteases are not much used in breadmaking in Europe.
The document EP021179 discloses the use of an alfa-amylase preparation in which the protease (inactivated) was used in combination with emulsifiers to inhibit staling.
Conforti et al. (1996) FSTA, 96(12), M0190 Abstract of presentation) added an enzyme mixture comprising bacterial amylase, fungal amylase and fungal protease to fat substituted muffins. The control fat containing muffin was more tender. The enzyme treatment decreased the staling rate. This is not surprising in view of the presence of amylases.
Lipase is also known to soften bread crumb and to somewhat reduce the firming rate of bread crumb (WO 94/04035 example 2).
Fungal proteases are sensitive to high temperatures. Their potency of protein hydrolysis in a moderate to high temperature range of about 50° C. or higher is normally poor. Some bacterial neutral and alkaline proteases are resistant to higher heat treatments. Till now reports on bacteria-derived proteases with heat resistance that can retain good peptidase activity, for example, in a high temperature range of about 60° C. have been scarce. The document EP1186658 discloses such enzyme produced by a bacterium of the genus Bacillus subtilis, more specifically an M2-4 strain. The disclosed enzyme mixture, however, completely looses its activity at a temperature of about 70° C. Neutral thermostable proteases from Bacillus, which may be tolerant to oxidising agents, are preferred in detergent formulations. Also alkaline thermostable proteases from Bacillus are used in washing and detergent formulations. Papain is very heat stable and requires a prolonged heating at 90-100° C. for deactivation. Bromelain is less stable and can be deactivated at around 70° C. Other heat stable proteases are produced by Bacillus licheniformis NS70 (Chemical Abstracts, 127, 4144 CA), Bacillus licheniformis MIR 29 (Chemical Abstracts, 116, 146805 CA), Bacillus stearothermophilus (Chemical Abstracts, 124, 224587 CA), Nocardiopsis (Chemical Abstracts, 114, 162444 CA) and Thermobacteroides (Chemical Abstracts, 116, 146805 CA). This is not an exhaustive list, but it illustrates the importance of the thermostable serine proteases and their application, mostly in detergents. No reference is made to baking and anti-staling properties.
Lipase is also known to soften bread crumb and to somewhat reduce the firming rate of bread crumb (WO 94/04035 example 2).
Fungal proteases are sensitive to high temperatures. Some bacterial neutral and alkaline proteases are resistant to higher heat treatments. Neutral thermostable proteases from Bacillus, which may be tolerant to oxidising agents, are preferred in detergent formulations. Also alkaline thermostable proteases from Bacillus are used in washing and detergent formulations. Papain is very heat stable and requires a prolonged heating at 90-100° C. for deactivation. Bromelain is less stable and can be deactivated at around 70° C. Other heat stable proteases are produced by Bacillus licheniformis NS70 (Chemical Abstracts, 127, 4144 CA), Bacillus licheniformis MIR 29 (Chemical Abstracts, 116, 146805 CA), Bacillus stearothermophilus (Chemical Abstracts, 124, 224587 CA), Nocardiopsis (Chemical Abstracts, 114, 162444 CA) and Thermobacteroides (Chemical Abstracts, 116, 146805 CA). This is not an exhaustive list, but it illustrates the importance of the thermostable serine proteases and their application, mostly in detergents. No reference is made to baking and anti-staling properties.
Papain is a proteolytically active constituent in the latex of the tropical papaya fruit. The crude dried latex contains a mixture of at least four cysteine proteinases.
Thermolysin is an extracellular, metalloendopeptidase secreted by the gram-positive thermophilic bacterium Bacillus thermoproteolyticus. 